The glomerular microvasculature is a common target of dysregulated or pathologic complement (C) activation. This has been implicated in the pathogenesis of a wide range of glomerulopathies including lupus nephritis, membranoproliferative glomerulopathy, postinfectious glomerulonephritis and, more recently, the atypical Hemolytic Uremic Syndrome (aHUS), and C3 glomerulopathy. Therefore, development of strategies to minimize activation of C cascades could be promising in C-depended glomerular diseases. The long-term goal of our research is to mitigate severity of C-mediated glomerular injury by up regulating expression of the decay-accelerating factor (DAF), a key controller of C activation acting by preventing assembly and accelerating decay of the naturally labile C3 and C5 convertases that amplify the classical and alternative complement activation cascades. As there are currently no pharmacologic DAF inducers, we sought to identify up regulators of DAF expression whose activity can be increased using existing pharmacologic strategies or agents. We identified Heme Oxygenase (HO)-1, the inducible enzyme of heme degradation to carbon monoxide (CO) and biliverdin, to be a DAF regulator in glomeruli. We demonstrated that HO-1 overexpression targeted to visceral glomerular epithelial cells (also known as podocytes) upregulates DAF and reduces C deposition and extent of injury. Owing to their non-replicative terminally differentiated nature, podocytes are particularly vulnerable to C-mediated injury while their loss was shown to be a key determinant of progression of glomerular diseases. The mechanism by which HO-1 up regulates DAF in podocytes is unknown. Also unknown is the extent to which DAF upregulation by HO-1 is critical in mitigating C-mediated podocyte injury. The proposed studies address these questions by pursuing the following Specific Aims: 1) To identify cis-acting positive response DAF promoter elements in rat podocytes over expressing HO-1, and examine the role of the HO reaction product, CO, as activator of specific DAF promoter transcription factors. This Aim will test the hypothesis that increased CO production in podocytes over expressing HO-1 up regulates DAF expression via CO responsive cis-acting positive regulatory elements on the DAF promoter corresponding to Sp1 transcription factor binding sites. 2) To demonstrate that increased DAF expression is a key mechanism by which HO-1 mitigates complement-dependent podocyte injury. This Aim will test the hypothesis that, in podocytes overexpressing HO-1, DAF mediates attenuation of C-dependent injury The experimental approach in Aim I includes use of cultured podocytes originating from Wild-Type (WT) rats and from rats lacking HO-1 we generated using zinc-finger nuclease (ZFN)-driven HO-1 gene knock- out. In these cells, the effect of HO-1 overexpression and of the HO reaction product, CO, on transcriptional regulation of DAF promoter via the Sp1 transcription factor will be assessed. The experimental approach in Aim II includes use of a rat model of antibody-mediated C-dependent podocyte injury resembling human membranous nephropathy, which will be induced in rats with podocyte- targeted HO-1 overexpression we generated using sleeping beauty (SB) transposon-mediated transgenesis that are either DAF replete or DAF deficient. Observations from the proposed studies are expected to have positive translational impact because the demonstration that HO-1 minimizes C-mediated injury via DAF induction could lead to novel strategies for therapeutic interventions in C-dependent renal diseases.